The overall objective of this application is to better understand the molecular basis of ligand-induced activation states of the secretin receptor, a prototypic class B G protein-coupled receptor. These insights should fill key gaps in current knowledge and facilitate the ultimate development of receptor-active drugs. This receptor family includes established targets for treatment of diabetes, obesity, osteoporosis, migraine, anxiety, and depression; however, development of small molecule agonists acting at these targets has been particularly challenging. Recently solved crystal structures demonstrating uniquely open helical bundle domains of two family members have helped to explain this challenge. This domain has, therefore, become the focal point of the current proposal, with component aims directed at the impact of helical bundle interactions with (i) biologically active ligands, (ii) th receptor amino terminus, and (iii) associated membrane proteins. The CENTRAL HYPOTHESIS is that conformational changes in this domain that initiate a spectrum of intracellular signaling events can be differentially affected by various ligands that interact with it directly, or indirecly by binding to the receptor amino terminus, and by interactions with other membrane proteins within quaternary complexes. New recognition of secretin receptor involvement in a unique cross-class hetero-receptor complex with the angiotensin 1a receptor provides insights relevant to physiology and therapeutics, and there is also strong rationale for use of a secretin receptor agonist to manage related major health problems, including obesity, diabetes, hypertension, and heart failure, as well as cholestasis. Our SPECIFIC AIMS will test three hypotheses: (i) the open space high in the helical bundle that is devoid of traditional docking pockets where the orthosteric agonist acts can be effectively mapped; (ii) amino-terminal and core domains interact in a specific and dynamic manner that affects the state of activation of the receptor; and (iii) key molecular interactions exist within a cross-class hetero-receptor complex that can be utilized to selectively modulate important biological activities. The first aim will explore the molecular determinants of agonist activity by utilizing cysteine trapping of residues at the top of transmembrane segments and within extracellular loops using cysteines in positions comprising the helix N-capping motif, and evaluating potential candidate pharmacophores directed to the natural activation pocket by attachment to a ligand that can be applied as a site-selective anchor that binds with high affinity to the receptor amino terminus. The second aim will gain insights into the receptor holo- structure by utilizing biochemical and mutagenesis strategies to define and disrupt or stabilize the interfaces between the secretin receptor amino terminus and core, and to examine functional implications. The third aim will explore the physical association between secretin and angiotensin 1a receptors, defining the structural basis and functional implications of their association, the impact of the full spectrum of types of drugs acting at each receptor, and developing strategies to take advantage of this unique hetero-receptor complex.